CN102051016B - Degradable amphiphilic triblock copolymer micelle and preparation method and application of degradable amphiphilic triblock copolymer micelle - Google Patents

Abstract

The invention relates to a degradable amphiphilic tri-block copolymer composed of polyethylene glycol segment, polycaprolactone segment and polyacrylic acid segment, and a degradable amphiphilic tri-block copolymer formed by self-assembly of the amphiphilic tri-block copolymer. A degradable amphiphilic triblock copolymer micelle, a preparation method of the degradable amphiphilic triblock copolymer and a preparation method of the degradable amphiphilic triblock copolymer micelle. The degradable amphiphilic tri-block copolymer micelle of the present invention is a degradable amphiphilic tri-block copolymer composed of a polyethylene glycol segment, a polycaprolactone segment and a polyacrylic acid segment. Assembled, the degradable amphiphilic triblock copolymer micelle is used as a drug carrier, and anti-tumor drugs are loaded on the carrier through hydrophobic interaction and coordination complexation to obtain anti-tumor nanoparticles. Particles can be used as antineoplastic agents.

Description

Degradable amphiphilic triblock copolymer micelle and its preparation method and use

technical field

The invention relates to a degradable amphiphilic tri-block copolymer composed of polyethylene glycol segment, polycaprolactone segment and polyacrylic acid segment and the degradable amphiphilic tri-block copolymer formed by self-assembly A micelle, a preparation method of a degradable amphiphilic triblock copolymer and a preparation method of a degradable amphiphilic triblock copolymer micelle. The present invention also relates to using the degradable amphiphilic triblock copolymer micelle as a drug carrier, loading anti-tumor drugs through hydrophobic interaction and coordination complexation to obtain anti-tumor nanoparticles, and using the anti-tumor nanoparticles For antitumor agents.

Background technique

Cancer is a disease that seriously threatens human health and safety. Affected by bad living habits and environmental pollution, the morbidity and mortality of cancer continue to rise, becoming the second leading cause of death in humans. Cancer treatment methods mainly include surgery, radiotherapy and chemotherapy. Among them, surgical treatment is traumatic to the human body and has high risk. It cannot remove scattered cancer cells, and has a strong recurrence rate. It may also cause tumor cells to spread and metastasize due to surgical stimulation. Forming an effective drug concentration or therapeutic dose locally in the tumor has poor effect and high toxicity, and simply increasing the drug or radiation dose is limited by systemic toxicity. Therefore, novel cancer treatment methods are urgently needed to be developed.

A new type of chemotherapy involving polymers is called Polymer Therapeutics. Among them, the polymer is used as a macromolecular prodrug (prodrug, which refers to a substance that releases a drug through a related reaction after entering the body), a drug or a protein carrier, and its size is 5-100 nanometers. Polymer therapy is currently successfully used in clinical treatment with polymer-loaded protein systems, and clinical trials of polymer-loaded anti-tumor drug systems are also progressing smoothly. The advantage of polymer therapy is that the size of the polymer as a prodrug or carrier is much larger than that of small-molecule anti-tumor drugs. In tumor tissue blood vessels where cells are loosely arranged due to rapid proliferation, macromolecules can enter tumor tissue through osmosis, while tumor The local lymphatic circulation function is weak, so macromolecules are not easy to be metabolized and excreted, and they are enriched in the tumor site. This effect of passive targeting behavior through the large molecular volume is called the EPR effect (the enhanced permeability and retention effect). In addition, active targeting can also be achieved by inserting specific targeting groups on the polymer to improve the targeting of the drug, so that the effective drug concentration or therapeutic dose can be achieved locally in the tumor, while the effect of the drug on other parts of the body can be reduced. Toxicity is minimized (1: K.L.Kiick, Science, Vol.317, 1182-1183 (2007); 2: R.Duncan, Nat.Rev.DrugDiscovery, Vol.2, 347-360 (2003); 3: H . Maeda et al. Adv PolymSci, Vol. 193, 103-121 (2005)).

Amphiphilic block copolymers can self-assemble into core-shell nanoparticles in aqueous solution, which can be used as drug carriers in polymer therapy. The shell of this nanoparticle is usually a hydrophilic block, which can effectively prevent protein adsorption and cell adhesion during in vivo circulation, and increase the circulation time in the blood so that more effective drugs can be transported to the tumor site without being overwhelmed. Early metabolism is excreted from the body, and can greatly reduce the renal toxicity caused by the metabolism of small molecule drugs and the toxicity of small molecule drugs to other organs due to non-targeting. Nanoparticle cores are usually hydrophobic blocks, which can be loaded with hydrophobic anti-tumor drugs through hydrophobic interactions, such as using block polymer micelles to load hydrophobic anti-tumor drugs doxorubicin or paclitaxel; achieve the purpose of drug controlled release, such as utilizing the carboxylate group of the two-block polymer polyethylene glycol-block-polyamino acid to realize the loading and slow release of cisplatin or diaminocyclohexane platinum ( 1: G.S.Kwon et al. Drug Development Research, Vol.67, 15-22 (2006); 2: G.-H.Hsiue et al. Advanced Functional Materials, Vol.17, 2291-2297 (2007); 3: Y.Y.Yang et al. Biomaterials , Vol.28, 1730-1740 (2007); 4: Y. Matsumura, K. Kataoka et al. British Journal of Cancer, Vol.93, 678-687 (2005); 5: H. Cabral, K. Kataoka et al. Journal of Controlled Release, Vol. 121, 146-155 (2007)).

Since single-agent chemotherapy often leads to increased resistance of tumor cells to drugs, the development of polymer therapy should be transformed from a single function to a multifunctional direction. This requires that the polymer used as the carrier has multifunctional characteristics, and can be loaded with the same or different antitumor drugs through different loading methods. The biocompatibility of the polymer carrier itself and the metabolic excretion after drug release are also the basic requirements for practical carriers.

Contents of the invention

One of the objectives of the present invention is to provide a degradable amphiphilic tri-block copolymer to obtain a degradable amphiphilic tri-block copolymer micelle through self-assembly of an aqueous solution in view of the single deficiency of the existing polymer carrier drug-loading function .

The second object of the present invention is to provide a preparation method of degradable amphiphilic triblock copolymer micelles.

The third object of the present invention is to provide a method for preparing a multifunctional degradable amphiphilic tri-block copolymer composed of polyethylene glycol segment, polycaprolactone segment and polyacrylic acid segment.

The fourth object of the present invention is to provide a degradable amphiphilic triblock copolymer composed of polyethylene glycol segment, polycaprolactone segment and polyacrylic acid segment through aqueous solution self-assembly. The use of the three-block copolymer micelle, the degradable amphiphilic three-block copolymer micelle is used as a drug carrier, and the anti-tumor drug is loaded on the carrier through hydrophobic interaction and coordination complexation to obtain an anti-tumor nanometer Particles, the anti-tumor nanoparticles can be used as anti-tumor agents.

The degradable amphiphilic triblock copolymer micelle of the present invention is a multifunctional degradable amphiphilic triblock copolymer composed of polyethylene glycol segment, polycaprolactone segment and polyacrylic acid segment Obtained after self-assembly in aqueous solution, the degradable amphiphilic triblock copolymer micelle has a core-shell structure, and its average particle diameter is 20-200 nanometers; wherein: the outer shell of the micelle is polyethylene glycol, and the inner core For polycaprolactone, polyacrylic acid is located on the outer surface of the inner core.

The degradable amphiphilic tri-block copolymer is composed of polyethylene glycol segment, polycaprolactone segment and polyacrylic acid segment, which has the following structural formula (A):

In the above formula, R ₁ is derived from the terminal structure of the polyethylene glycol segment, and R ₁ is a hydrogen atom, carboxyl group, amino group, cyano group, mercapto group, formyl group, aldehyde group, or a linear or branched C _1- One of ₁₂ alkyl groups; L is a linking group, which is derived from one of halogenated carboxylic acid, halogenated acid chloride or halogenated acid bromide and polyethylene glycol-block-polycaprolactone Residue after reaction of segment copolymer, said residue is preferably -CO-C(CH ₃ ) ₂ -, -CO-CH(CH ₃ )-, -CO-C ₆ H ₄ -CH ₂ - or -CO -CH(CN)-; R ₂ is a halogen, which is a diblock copolymer of a halogenated carboxylic acid, a halogenated acid chloride or a halogenated acid bromide and a polyethylene glycol-block-polycaprolactone Obtained after the reaction; m is an integer of 45-120, n is an integer of 5-60, y is an integer of 10-100; x is the hydrolysis rate, and the hydrolysis rate is 30%-100%, preferably 70%-100%, More preferably, it is 100%.

The halogen is Cl, Br or I.

Described C _1-12 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, decyl or undecyl wait.

When the hydrolysis rate x is 100%, the polyacrylic acid chain segment is hydrolyzed completely, and the side chain is completely carboxyl; when 30%≤x<100%, the polyacrylic acid chain segment is hydrolyzed incompletely, and the carboxyl group of the side chain has no tert-butyl ester group. regular arrangement.

The degradable amphiphilic tri-block copolymer micelle of the present invention can be prepared by the following method: the degradable amphiphilic tri-block copolymer is dissolved in an organic solvent (the amount of the organic solvent is to completely dissolve the degradable amphiphilic Three-block copolymer is advisable, preferably degradable amphiphilic three-block copolymer is in organic solvent mass concentration is 1-20g/L), obtains the solution that contains degradable amphiphilic three-block copolymer, will The solution containing the degradable amphiphilic tri-block copolymer is dropped into water, or water is dropped into the solution containing the degradable amphiphilic tri-block copolymer, wherein the volume ratio of water to organic solvent is greater than 1, preferably water and organic solvent 1<volume ratio≤100, more preferably the volume ratio of water to organic solvent is 10-100; then the organic solvent is removed by ultrafiltration or dialysis to obtain degradable amphiphilic triblock copolymer micelles. Among them, the organic solvent is preferably an organic solvent having good solubility for the degradable amphiphilic triblock copolymer, for example, at least one selected from the group consisting of tetrahydrofuran, acetone, dimethylformamide and the like. The formed degradable amphiphilic triblock copolymer micelles usually have good dispersion, and their average particle diameter is 20-200 nanometers.

The degradable amphiphilic triblock copolymer of the present invention can be prepared by the following method:

(1) Mix polyethylene glycol (PEG) with R ₁ at one end and hydroxyl at the other end and caprolactone (CL) monomer at a molar ratio of 1:5-200, and then dissolve the mixture as a reactant In dry toluene (the amount of toluene is advisable to completely dissolve the reactant, the mass concentration of the preferred reactant in toluene is 50-200g/L), add catalyst amount of 1,5,7-triazabicyclo-[4.4.0 ]dec-5-ene (TBD), react at room temperature, and the reaction time is controlled according to the required degree of polymerization (generally 1-200 hours); or

The mixture is used as the reactant, and the catalyst stannous octoate is added in the reactant, wherein the addition of the stannous octoate is that the molar ratio of PEG to stannous octoate is 10-30; at a reaction temperature of 100-150 ° C, the reaction (preferably The reaction temperature is 120°C), and the reaction time is controlled according to the required degree of polymerization (generally 1-200 hours);

The product obtained by the reaction is precipitated and dried in a precipitating agent (precipitating agent is selected from ether, normal hexane, petroleum ether) to obtain polyethylene glycol-block-polycaprolactone diblock copolymer (PEG _m- PCL _n -OH, m is an integer of 45-120, and n is an integer of 5-60), wherein the terminal group of polyethylene glycol chain segment is R ₁ (the definition of R ₁ is the same as above), polycaprolactone chain The end group of the segment is a hydroxyl group;

(2) The end group of the polyethylene glycol chain segment obtained in step (1) is R ₁ , and the end group of the polycaprolactone chain segment is a polyethylene glycol-block-polycaprolactone two-block The copolymer and the halide are mixed in a molar ratio of 1:1-10, and then the mixture is dissolved in dry dichloromethane as a reactant (the amount of dichloromethane is preferably to completely dissolve the reactant, and the preferred reactant is in dichloromethane The mass concentration in methane is 50-200g/L), adding dry triethylamine (TEA) as a catalyst, wherein the molar ratio of triethylamine to halide is 1, react at room temperature for 2-48 hours, and remove by filtration After the salt generated by the reaction, precipitate and dry to obtain polyethylene glycol-block-polycaprolactone diblock copolymer initiator ( PEG _m -PCL _n -X, m is an integer of 45-120, and n is an integer of 5-60), wherein the terminal group of polyethylene glycol segment is R ₁ (the definition of R ₁ is the same as above), polyethylene glycol The end group of the lactone segment is a halogen (X), and the halogen is Cl, Br or I;

The halogenated compound is selected from α-halogenated isobutyric acid, α-halogenated isobutyryl chloride, α-halogenated isobutyryl bromide, α-halogenated isopropionic acid, α-halogenated isopropionyl chloride, α- Halogenated isopropionyl bromide, Halogenated methylbenzoic acid, Halogenated methylbenzoyl chloride, Halogenated methylbenzoyl bromide, α-halogenated cyanoacetic acid, α-halogenated cyanoacetyl chloride, α-halogenated cyanoacetyl bromide one of

(3) The end group of the polyethylene glycol segment obtained in step (2) is R ₁ , and the end group of the polycaprolactone segment is a polyethylene glycol-block-polycaprolactone diblock The copolymer initiator is mixed with tert-butyl acrylate (tBA) monomer in a molar ratio of 1: 10-300, and then the mixture is dissolved in dry anisole or acetone as a reactant (the amount of anisole or acetone The amount is advisable to completely dissolve the reactant, preferably the mass concentration of the reactant in anisole or acetone is 50-2000g/L), add ketone bromide (CuBr) and pentamethyldiethylenetriamine (PMDETA ), wherein polyethylene glycol-block-polycaprolactone diblock copolymer initiator: the molar ratio of CuBr: PMDETA is 2: 1: 1, any constant temperature between room temperature and 120 ℃, and Reaction under anaerobic conditions; the reaction time is controlled according to the required degree of polymerization (generally 1-200 hours); the product obtained by the reaction is diluted with dichloromethane solvent (dichloromethane is for the purpose of diluting the product, and the amount added is not limited. , the mass concentration of the preferred product in dichloromethane is 2-3g/L) and after the perbasic aluminum oxide column, the solvent dichloromethane is removed by rotary evaporation. Precipitate in and dry to obtain polyethylene glycol-block-polycaprolactone-block-polyacrylate tert-butyl ester triblock copolymer (PEG _m -PCL _n -PtBA _y , m is 45-120 integer, n is an integer of 5-60, and y is an integer of 10-100), wherein the end group of the polyethylene glycol segment is R ₁ (the definition of R ₁ is the same as above), and the poly(tert-butyl acrylate) segment The terminal group of is a halogen, and the halogen is Cl, Br or I;

(4) the polyethylene glycol-block-polycaprolactone-block-polyacrylate tert-butyl ester triblock copolymer that step (3) obtains is dissolved in methylene chloride (the amount of methylene chloride is based on complete It is suitable to dissolve polyethylene glycol-block-polycaprolactone-block-polyacrylate tert-butyl ester triblock copolymer, preferably polyethylene glycol-block-polycaprolactone-block-polyacrylate tertiary The mass concentration of butyl ester triblock copolymer in dichloromethane is 10-200g/L), adding trifluoroacetic acid (TFA) as a catalyst, wherein TFA and polyethylene glycol-block-polycaprolactone-block The molar ratio of the tert-butyl group of the poly-tert-butyl acrylate in the section-polyacrylate tert-butyl ester triblock copolymer is 3-10, reacts 10-200 hour at room temperature, the gained product after the reaction is in precipitation agent (precipitation agent selected from diethyl ether, n-hexane, petroleum ether) and dried to obtain the degradable amphiphilic triblock copolymer polyethylene glycol-block-polycaprolactone-block copolymer of the above formula (A) Segment - polyacrylic acid [PEG _m -PCL _n -P(AA _x -tBA _1-x ) _y ]. Among them, m is an integer of 45-120, n is an integer of 5-60, y is an integer of 10-100, x represents the hydrolysis rate, when x is 100%, the polyacrylic acid segment is hydrolyzed completely, and the side chain is completely carboxyl ; When 30%≤x<100%, the hydrolysis of the polyacrylic acid segment is not complete, and the carboxyl group and the tert-butyl ester group of the side chain are randomly arranged. The end group of the polyethylene glycol chain segment is R ₁ (the definition of R ₁ is the same as above), and the end group of the polyacrylic acid chain segment is halogen, and the halogen is Cl, Br or I.

The three segments of the degradable amphiphilic tri-block copolymer can be connected in any way, and as long as it is for the purpose of the present invention, they can be combined with any linking group. The production method is not particularly limited. In addition to the above method, the degradable amphiphilic tri-block copolymer of the present invention can also be prepared by first synthesizing three segments respectively, and then covalently connecting the three segments through terminal group reactions. The substance prepared by the above method has basically the same structure, and the difference may be that the linking group is a structure after the reactive group is covalently connected.

The degradable amphiphilic triblock copolymer micelle described in the present invention can be used as a drug carrier.

The drug carrier is to load hydrophobic antitumor drugs through the hydrophobic interaction of the hydrophobic biodegradable polycaprolactone segment of the degradable amphiphilic tri-block copolymer, and then through the carboxylate group of the polyacrylic acid segment Coordination and complexation with platinum in cis-platinum (II) anti-tumor drugs Loading cis-platinum (II) (II represents divalent platinum) anti-tumor drugs to form anti-tumor nanoparticles. The anti-tumor nanoparticles have a core-shell structure with an average particle size of 20-200 nanometers; wherein: the outer shell of the anti-tumor nanoparticles is polyethylene glycol; the inner core is polyhexene loaded with hydrophobic anti-tumor drugs. ester; the polyacrylic acid loaded with cis-platinum (II) antineoplastic drugs through coordination complexation is located on the outer surface of the inner core.

In the present invention, cis-platinum (II) anti-tumor drugs can also be loaded to form anti-tumor nanoparticles through the coordination and complexation of the carboxylate radical of the polyacrylic acid chain segment and the platinum in the cis-platinum (II) anti-tumor drugs, Then, hydrophobic antitumor drugs are loaded through the hydrophobic interaction of the hydrophobic biodegradable polycaprolactone segment of the degradable amphiphilic triblock copolymer to form antitumor nanoparticles.

The mass of the hydrophobic anti-tumor drug loaded on the anti-tumor nanoparticles accounts for 1%-50% of the mass of the anti-tumor nanoparticles; the cis-platinum (II) class anti-tumor drugs loaded on the anti-tumor nanoparticles The molar ratio (Pt/COO ⁻ ) of platinum (Pt) relative to carboxylate groups of the degradable amphiphilic triblock copolymer is 0.05-1, preferably 0.3-1.

The preparation process of the hydrophobic effect loaded hydrophobic antitumor drug of the polycaprolactone segment is to dissolve the degradable amphiphilic triblock copolymer and the hydrophobic antitumor drug in the organic solvent simultaneously (the degradable amphiphilic There is no specific restriction on the feed ratio of the amphiphilic triblock copolymer and the hydrophobic antitumor drug, and the mass ratio of the degradable amphiphilic triblock copolymer to the hydrophobic antitumor drug is 1-10, and the amount of the organic solvent is in the range of It is advisable to completely dissolve the degradable amphiphilic tri-block copolymer and the hydrophobic anti-tumor drug, preferably the total mass concentration of the degradable amphiphilic tri-block copolymer and the hydrophobic anti-tumor drug in the organic solvent is 1-20g /L) to obtain a mixed solution containing degradable amphiphilic tri-block copolymers and hydrophobic anti-tumor drugs, drop the mixed solution containing degradable amphiphilic tri-block copolymers and hydrophobic anti-tumor drugs water, or drop water into a mixed solution containing a degradable amphiphilic triblock copolymer and a hydrophobic antineoplastic drug, wherein the volume ratio of water to organic solvent is greater than 1, preferably water to organic solvent 1<volume ratio≤ 100, more preferably the volume ratio of water to organic solvent is 10-100; then the organic solvent and unloaded hydrophobic antitumor drug are removed by ultrafiltration or dialysis to obtain antitumor nanoparticles loaded with hydrophobic antitumor drug intermediate product; wherein, the organic solvent is preferably an organic solvent with good solubility for degradable amphiphilic triblock copolymers and hydrophobic antitumor drugs, for example, selected from the group consisting of tetrahydrofuran, acetone, dimethylformamide, etc. at least one of the

The preparation process of loading cis-platinum (II) anti-tumor drugs through the coordination and complexation of the carboxylate of the polyacrylic acid segment and the platinum in the cis-platinum (II) anti-tumor drugs is that the above-mentioned loaded The intermediate product of anti-tumor nanoparticles with hydrophobic anti-tumor drugs and cis-platinum (II) anti-tumor drugs are placed in water as reactants, wherein: platinum in cis-platinum (II) anti-tumor drugs is relative to polyacrylic acid The feeding molar ratio of the carboxylate group in the chain segment is 0.1-2; the amount of water is suitable for dissolving the reactant, preferably the mass concentration of the reactant is 1-20g/L, at any constant temperature between 20-50°C React for 24-96 hours, and then remove unloaded cis-platinum (II) antitumor drugs by ultrafiltration or dialysis to obtain antitumor nanoparticles.

The described cis-platinum (II) class antineoplastic drug can be reacted with silver nitrate in water at room temperature and protected from light (the molar ratio of Pt to silver nitrate is 1) for at least 12 hours, and then filtered to remove silver chloride to obtain hydrated cis-platinum (II). Platinum(II) contributes to the above-mentioned coordination complexation reaction.

Described hydrophobic antitumor drug, as long as it is for the purpose of the present invention, can be selected from any hydrophobic antitumor drug, preferably a hydrophobic antitumor drug that has been proved to have a good antitumor effect in laboratory and clinical experiments, for example, it can be selected from doxorubicin, epirubicin, daunorubicin, paclitaxel, camptothecin, 10-hydroxycamptothecin, 5-aminocamptothecin, vinblastine, vincristine, etoposide, cisplatin, At least one selected from the group consisting of carboplatin, oxaliplatin and the like.

The cis-platinum (II) class antineoplastic drug is a platinum compound, selected from cisplatin, carboplatin, cycloplatin, heptaplatin, denaplatin, cyclopentylamidoplatinum, platinum blue, cyproplatinum, ethylenedi Amine malonate platinum, niniplatin, enloplatin, epithioplatin, cispiroplatin, dextromethopretin, isoproplatin, lobaplatin, rice platinum, picoplatin, nedaplatin, omaplatin, oxaliplatin, At least one of the group consisting of spatioplatin, spiroplatin, sulplatin, dicycloplatin, eberplatin, meciplatin, cisliplatin, picoplatin, geniplatin and the like.

R ₁ is derived from the terminal structure of the polyethylene glycol chain segment. When R ₁ is carboxyl, amino, cyano, mercapto, formyl or aldehyde group, it can be used as follows: for example, in forming the antitumor nanometer of the present invention After the particles are prepared, the above-mentioned groups are covalently bonded to appropriate antibodies or fragments with specific binding properties (such as glycosyl, folic acid, cRGD) as needed, so that the anti-tumor nanoparticles can actively target tumor tissues.

The polyethylene glycol segment in the anti-tumor nanoparticles of the present invention is a hydrophilic segment as the outer shell, which acts to stabilize the anti-tumor nanoparticles and prevent protein adsorption and cell adhesion when they are used in vivo, while increasing Circulation time of antitumor nanoparticles in vivo. Polyethylene glycol is a polymer that has no toxic side effects on patients (mammals, especially humans), can be excreted by metabolism, and is widely used in polymer carriers or prodrugs in polymer therapy. As a hydrophobic segment, the polycaprolactone segment has dual functions: first, anti-tumor nanoparticles require the polycaprolactone segment to load hydrophobic anti-tumor drugs through hydrophobic interactions, and the drugs are released through the diffusion mechanism after entering the patient's body , when the polycaprolactone segment is degraded under the action of enzymes, the drug is completely released with the disintegration of the anti-tumor nanoparticles; It can be degraded and broken under the physiological conditions of patients (mammals, especially humans), which helps the disintegration of anti-tumor nanoparticles and makes them easily excreted through metabolism. The polyacrylic acid segment is hydrophilic and has good solubility in alkaline aqueous solution, but poor solubility in neutral aqueous solution, so it is located on the outer surface of the anti-tumor nanoparticle core. The carboxylate group of the polyacrylic acid chain segment forms a coordination complex with the cis-platinum (II) anti-tumor drug, and the cis-platinum (II) anti-tumor drug is loaded in the anti-tumor nanoparticle. At least one of the 2 chlorine groups of cis-platinum (II) is replaced by the carboxylate on the side chain of the polyacrylic acid segment in the degradable amphiphilic triblock copolymer, and the remaining chlorine groups are hydrated (referring to water The molecule is coordinated in the platinum state by the lone electron pair of its oxygen atom). When two of the above-mentioned chlorine groups are replaced by two carboxylate groups, the two carboxylate groups can be derived from adjacent or separated carboxylate groups of a single degradable amphiphilic triblock copolymer molecule, or derived from The plurality of degradable amphiphilic triblock copolymer molecules is not particularly limited.

As long as it is for the purpose of the present invention, the carboxylate group contained in the polyacrylic acid segment in the anti-tumor nanoparticles can also be used to load other drugs or auxiliary drugs related to tumor treatment through coordination and complexation, and there is no special limitation. The radioactive isotopes ⁹⁰ Y, ⁶⁰ Co, and ⁶⁷ Cu for internal radiation therapy of tumors, Gd(III) for in vivo magnetic resonance imaging (MRI) and superparamagnetic iron oxide particles (SPIO) for dynamic enhanced MR imaging are listed. ).

There is no specific limitation on the order of the two loading methods of loading the same or different types of anti-tumor drugs through hydrophobic interaction and coordination complexation in the preparation of the anti-tumor nanoparticles described in the present invention. Preferably, the hydrophobic antitumor drug is firstly loaded through hydrophobic interaction to form nanoparticles, and then the cis-platinum (II) antitumor drug is loaded through a coordination complexation reaction.

The solution containing the anti-tumor nanoparticles prepared above can be directly sterilized as it is, and as needed, it can be prepared as an injection by adding its own known adjuvant suitable for injection; or after the solution containing the anti-tumor nanoparticles is concentrated, for example, carry out Freeze-dried to make a solid micropowder, which can be redissolved in an injectable solution, and can be mixed with a pharmaceutically acceptable carrier made into a certain shape, and processed into a drug suitable for various administrations. form of dosage form. Representative examples of such carriers can be deionized water, aqueous solutions buffered at a certain pH value, monosaccharides or oligosaccharides, sugar alcohols, etc., but are preferably used as suitable for parenteral administration, especially intravenous or subcutaneous administration The compositions are provided in dosage forms.

The anti-tumor drugs loaded on the anti-tumor nanoparticles of the present invention may be any combination of anti-tumor drugs as long as they do not produce substances that adversely affect the efficacy of the drugs when loaded together.

The anti-tumor nanoparticles of the present invention can be used in combination with other anti-tumor agents. These antitumor agents may be any agents as long as they do not cause adverse effects when used in combination, such as cytarabine, 5-FU, adriamycin, paclitaxel, camptothecin, cisplatin, and the like. These combined drugs may be administered by the same or different routes of administration of two or more drugs combined at the same time or at different times.

Description of drawings

Figure 1 is the NMR spectrum of the degradable amphiphilic triblock copolymer A described in Example 1 of the present invention.

Fig. 2 is the NMR spectrum of the degradable amphiphilic triblock copolymer B described in Example 2 of the present invention.

Fig. 3 is the NMR spectrum of the degradable amphiphilic triblock copolymer C described in Example 3 of the present invention.

Fig. 4 is the NMR spectrum of the degradable amphiphilic triblock copolymer D described in Example 4 of the present invention.

Fig. 5 is a schematic structural diagram of the degradable amphiphilic triblock copolymer micelle described in Example 5 of the present invention.

Fig. 6 is the radius distribution curve of the degradable amphiphilic triblock copolymer micelles given by the dynamic light scattering described in Example 5 of the present invention. Fig. 6 (A) is the radius distribution curve of degradable amphiphilic triblock copolymer micelle A; Fig. 6 (B) is the radius distribution curve of degradable amphiphilic triblock copolymer micelle B; Fig. 6 (C) is the radius distribution curve of the degradable amphiphilic triblock copolymer micelle C; FIG. 6(D) is the radius distribution curve of the degradable amphiphilic triblock copolymer micelle D.

Fig. 7 is a schematic diagram of the structure of the anti-tumor nanoparticles described in Example 6 of the present invention.

Fig. 8 is a radius distribution curve of anti-tumor nanoparticles obtained by dynamic light scattering described in Example 6 of the present invention. Fig. 8 (A) is the radius distribution curve of anti-tumor nanoparticles A; Fig. 8 (B) is the radius distribution curve of anti-tumor nanoparticles B; Fig. 8 (C) is the radius distribution curve of anti-tumor nanoparticles C; Fig. 8 (D) is the radius distribution curve of anti-tumor nanoparticles D.

Fig. 9 is the antitumor drug release curve of the antitumor nanoparticle A described in Example 7 of the present invention in 10mM PBS (pH7.4) buffer solution containing 160mM NaCl. Figure 9(A) is the release curve of doxorubicin, and Figure 9(B) is the release curve of cisplatin.

Fig. 10 is a diagram of the degradation behavior of the degradable amphiphilic triblock copolymer micelle A described in Example 8 of the present invention in an aqueous solution in the presence of lipase Lipase PS.

Detailed ways

The present invention is specifically described below through specific examples, but the present invention is not limited by these specific examples.

Embodiment 1: Synthesis of degradable amphiphilic triblock copolymer A

The structural formula of the degradable amphiphilic triblock copolymer A synthesized in this embodiment is as follows:

(1.a.) 10.0g polyethylene glycol (PEG, one end is methoxy group, one end is hydroxyl group, number average molecular weight is 5000, 2mmol) and 9.1g caprolactone (CL, molecular weight is 114, 80mmol) is dissolved In 125mL of dry toluene, add 30mg TBD (1,5,7-triazabicyclo-[4.4.0]dec-5-ene) as a catalyst, react at room temperature for 22 hours, precipitate in 400mL ether and Drying under high temperature gave 15.9 g of white powdery product (PEG ₁₁₄ -PCL ₂₈ -OH, number average molecular weight: 8200), with a yield of 83% and a monomer conversion rate of CL of 70%.

(1.b.) Dissolve 14.8 g of the above (1.a.) product (number average molecular weight 8200, 1.8 mmol) and 1.2 g bromoisobutyryl bromide (molecular weight 230, 5.4 mmol) in 100 mL of dry di In methyl chloride, use 0.75 mL of dry triethylamine (TEA, molecular weight: 101, 5.4 mmol) as a catalyst, react at room temperature for 48 hours, filter to remove the salt generated by the reaction, precipitate in 400 mL of ether and dry under vacuum 14.1 g of a white powdery product (PEG ₁₁₄ -PCL ₂₈ -Br, number average molecular weight: 8300) was obtained with a yield of 93%.

(1.c.) 0.2g of the above (1.b.) product (number average molecular weight 8300, 0.024mmol), 0.4g tert-butyl acrylate (tBA, molecular weight 128, 3.1mmol), 1.7mg CuBr (molecular weight 143, 0.012mmol), 2.5μL PMDETA (molecular weight: 173, 0.012mmol) were dissolved in 0.5mL anisole, reacted under anaerobic conditions at 120°C for 46 hours, added 300mL dichloromethane to dilute and perbasic alumina The solvent dichloromethane was removed by post-column rotary evaporation, precipitated in 400 mL of ether and dried under vacuum to obtain 0.23 g of a white solid product (PEG ₁₁₄ -PCL ₂₈ -PtBA ₂₅ , number average molecular weight 11500), with a yield of 33%. The monomer conversion of tBA was 19%.

(1.d.) 0.23g of the above (1.c.) product (number average molecular weight is 11500, 0.02mmol, wherein, tert-butyl is 0.5mmol) and 0.57g trifluoroacetic acid (TFA, molecular weight is 114, 5mmol ) was dissolved in 5mL of dichloromethane, reacted at room temperature for 120 hours, precipitated in 400mL of ether and dried under vacuum to obtain 0.2g of a white solid product, which was the degradable amphiphilic triblock copolymer A ( PEG ₁₁₄ -PCL ₂₈ -PAA ₂₅ , the number average molecular weight is 10100), the hydrolysis rate is 100%, and the yield is 100%. Fig. 1 is the NMR spectrum of degradable amphiphilic triblock copolymer A. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 1.30(m, 56H, -CH ₂ CH ₂ CH ₂ -derived from polycaprolactone), 1.55(m, 162H, -CH ₂ CH ₂ CH ₂ -derived In polycaprolactone, -CH ₂ CH(COO-)-derived from polyacrylic acid), 2.27(m, 81H, -CH ₂ C(=O)O-derived from polycaprolactone, -CH ₂ CH(COO -)-derived from polyacrylic acid), 3.51(s, 456H, -OCH ₂ CH ₂ O-derived from polyethylene glycol), 3.98(t, 56H, -C(=O)OCH ₂ -derived from polyhexanol ester), 12.25. (s, 25H, _-CH2CH (COOH)- derived from polyacrylic acid).

Example 2: Synthesis of degradable amphiphilic triblock copolymer B

The structural formula of the degradable amphiphilic triblock copolymer B synthesized in this embodiment is as follows:

(2.a.) 10.0g PEG (one end is methoxy group, one end is hydroxyl group number average molecular weight is 2000, 5mmol), 11.4g CL (100mmol) and 69mg stannous octoate (molecular weight is 405, 0.17mmol) are catalysts , reacted under anaerobic conditions at 120°C for 24 hours, precipitated in 400mL petroleum ether and dried under vacuum to obtain 16.3g of white powdery product (PEG ₄₅ -PCL ₈ -OH, number average molecular weight 2900), the yield was 76%, and the monomer conversion of CL was 40%.

(2.b.) Dissolve 8.7g of the above (2.a.) product (number average molecular weight: 2900, 3mmol) and 1.4g of bromoisobutyryl bromide (6mmol) in 100mL of dry dichloromethane, Dry TEA (6mmol) was used as a catalyst, reacted at room temperature for 2 hours, precipitated in 400mL petroleum ether and dried under vacuum to obtain a white powder product (PEG ₄₅ -PCL ₈ -Br, several The average molecular weight is 3100) 8.1 g, and the yield is 93%.

(2.c.) 0.17g of the above (2.b.) product (number average molecular weight 3100, 0.06mmol), 0.77g tBA (6mmol), 4.3mg CuBr (0.03mmol), 6.2μL PMDETA (0.03mmol) Dissolve in 3mL of acetone, react under anaerobic conditions at 60°C for 80 hours, add 400mL of dichloromethane to dilute and perbasic alumina column to remove the solvent, dichloromethane, precipitate in 400mL of petroleum ether and under vacuum conditions Drying gave 0.4 g of a white solid product (PEG ₄₅ -PCL ₈ -PtBA ₃₀ , number average molecular weight: 6900), with a yield of 43%, and a monomer conversion rate of tBA of 30%.

(2.d.) Dissolve 0.4g of the above (2.c.) product (number average molecular weight: 6900, 0.06mmol, wherein, tert-butyl is 1.8mmol) and 1.2g TFA (10.2mmol) in 8mL dichloromethane , reacted at room temperature for 96 hours, precipitated in 400 mL of petroleum ether and dried under vacuum to obtain a white solid product [PEG ₄₅ -PCL ₈ -P(AA _83% -tBA _17% ) ₃₀ , number average molecular weight 5500] 0.3 g is the degradable amphiphilic triblock copolymer B. The hydrolysis rate was 83%, and the yield was 93%. Figure 2 is the NMR spectrum of the degradable amphiphilic triblock copolymer B. ¹ H NMR (400MHz, DMSO-d ₆ ) δ1.29 (m, 16H, -CH ₂ CH ₂ CH ₂ -derived from polycaprolactone), 1.37(s, 45H, -OC ₄ H ₉ derived from polyacrylic acid ), 1.49-1.56 (m, 92H, -CH ₂ CH ₂ CH ₂ -derived from polycaprolactone, -CH ₂ CH(COO-)-derived from polyacrylic acid), 2.27 (m, 46H, -CH ₂ C (=O)O-derived from polycaprolactone, _-CH2CH (COO- ₎ -derived from polyacrylic acid), 3.51 (s, 180H, _-OCH2CH2O -derived from polyethylene glycol), 3.98 (t, 16H, -C(=O) _OCH2 -derived from polycaprolactone), 12.24. (s, 25H, _-CH2CH (COOH)-derived from polyacrylic acid).

Example 3: Synthesis of degradable amphiphilic triblock copolymer C

The structural formula of the degradable amphiphilic triblock copolymer C synthesized in this embodiment is as follows:

(3.a.) Dissolve 10.0g PEG (one end is methoxy group, one end is hydroxyl group, number average molecular weight is 5000, 2mmol) and 9.1g CL (80mmol) are dissolved in 125mL dry toluene, with 30mgTBD as catalyst, in Reacted at room temperature for 22 hours, precipitated in 400 mL of n-hexane and dried under vacuum to obtain 15.9 g of a white powder product (PEG ₁₁₄ -PCL ₂₈ -OH, number average molecular weight 8200), with a yield of 83%. The body conversion rate is 70%.

(3.b.) Dissolve 14.8g of the above (3.a.) product (number average molecular weight: 8200, 1.8mmol) and 1.2g of bromoisobutyryl bromide (5.4mmol) in 100mL of dry dichloromethane to 0.75mL of dry TEA (5.4mmol) was used as a catalyst, reacted at room temperature for 28 hours, filtered to remove the salt generated by the reaction, precipitated in 400mL of n-hexane and dried under vacuum to obtain a white powder product (PEG ₁₁₄ -PCL ₂₈ - Br, the number average molecular weight is 8300) 14.1g, and the productive rate is 93%.

(3.c.) Add 0.4g of the above (3.b.) product (number average molecular weight: 8300, 0.05mmol), 0.3gtBA (2.5mmol), 3.6mg CuBr (0.025mmol), 5.2μL PMDETA (0.025mmol) Dissolve in 0.5mL anisole, react under anaerobic conditions at 120°C for 48 hours, add 350mL dichloromethane to dilute and perbasic alumina column to remove solvent dichloromethane, precipitate in 400mL n-hexane and Drying under vacuum gave 0.3 g of a white solid product (PEG ₁₁₄ -PCL ₂₈ -PtBA ₁₁ , number average molecular weight: 9700), with a yield of 43% and a monomer conversion rate of tBA of 22%.

(3.d.) Dissolve 0.2 g of the above (3.c.) product (number average molecular weight: 9700, 0.02 mmol, wherein tert-butyl is 0.2 mmol) and 0.1 g TFA (1.0 mmol) in 3 mL of dichloromethane , reacted at room temperature for 72 hours, precipitated in 400 mL of n-hexane and dried under vacuum to obtain a white solid product [PEG ₁₁₄ -PCL ₂₈ -P(AA _70% -tBA _30% ) 11, number average molecular weight 9300] 0.18 g is the degradable amphiphilic tri-block copolymer C, with a hydrolysis rate of 70% and a yield of 98%. Figure 3 is the NMR spectrum of the degradable amphiphilic triblock copolymer C. ¹ H NMR (400MHz, DMSO-d ₆ ) δ1.24(m, 56H, -CH ₂ CH ₂ CH ₂ -derived from polycaprolactone), 1.29(s, 27H, -OC ₄ H ₉ derived from polyacrylic acid ), 1.54(m, 78H, -CH ₂ CH ₂ CH ₂ -derived from polycaprolactone, -CH ₂ CH(COO-)-derived from polyacrylic acid), 2.27(m, 67H, -CH ₂ C(= O)O-derived from polycaprolactone, _-CH2CH (COO- ₎ -derived from polyacrylic acid), 3.51(s, 456H, _-OCH2CH2O -derived from polyethylene glycol), 3.98(t , 56H, -C(=O) _OCH2 -derived from polycaprolactone), 12.26. (s, 8H, _-CH2CH (COOH)-derived from polyacrylic acid).

Example 4: Synthesis of degradable amphiphilic triblock copolymer D

The structural formula of the degradable amphiphilic triblock copolymer D synthesized in this embodiment is as follows:

(4.a.) 10.0g PEG (one end is methoxy group, one end is hydroxyl group number average molecular weight is 5000, 2mmol), 45.6g CL (400mmol) and 81mg stannous octoate (molecular weight is 405, 0.2mmol) as catalyst , reacted under anaerobic conditions at 120 °C for 48 hours, precipitated in 400 mL of petroleum ether and dried under vacuum to obtain 20.2 g of a white powdery product (PEG ₁₁₄ -PCL ₅₇ -OH, number average molecular weight 11500), with a yield of 36%, and the monomer conversion of CL was 29%.

(4.b.) Dissolve 11.5g of the above (4.a.) product (number-average molecular weight: 11500, 1mmol) and 2.3g of bromoisobutyryl bromide (10mmol) in 100mL of dry dichloromethane. Dry TEA (10mmol) was used as a catalyst, reacted at room temperature for 10 hours, precipitated in 400mL petroleum ether and dried under vacuum to obtain a white powder product (PEG _114- PCL ₅₇ -Br, several The average molecular weight is 11600) 10.9 g, and the yield is 93%.

(4.c.) 0.7g of the above (4.b.) product (number average molecular weight 11600, 0.06mmol), 2.3g tBA (18mmol), 4.3mg CuBr (0.03mmol), 6.2μL PMDETA (0.03mmol) Dissolve in 3mL of acetone, react for 120 hours at room temperature under anaerobic conditions, add 1000mL of dichloromethane to dilute and perbasic alumina column, spin evaporate to remove solvent dichloromethane, precipitate in 400mL of petroleum ether and dry under vacuum 1.4 g of a white solid product (PEG ₁₁₄ -PCL ₅₇ -PtBA ₁₀₀ , number average molecular weight: 24400) was obtained, with a yield of 47% and a monomer conversion rate of tBA of 33%.

(4.d.) Dissolve 1.2g of the above (4.c.) product (number average molecular weight is 24400, 0.05mmol, wherein the tert-butyl group is 5mmol) and 1.7gTFA (15mmol) in 20mL dichloromethane, at room temperature reacted at low temperature for 24 hours, precipitated in 400 mL of petroleum ether and dried under vacuum to obtain 1.1 g of a white solid product [PEG ₁₁₄ -PCL ₅₇ -P(AA _30% -tBA _70% ) ₁₀₀ , number average molecular weight 22800], namely is the degradable amphiphilic triblock copolymer D. The hydrolysis rate is 30%, and the yield is 93%. Figure 4 is the NMR spectrum of the degradable amphiphilic triblock copolymer D. ¹ H NMR (400MHz, DMSO-d ₆ ) δ1.29(m, 114H, -CH ₂ CH ₂ CH ₂ -derived from polycaprolactone), 1.39(s, 630H, -OC ₄ H ₉ derived from polyacrylic acid ), 1.49-1.57 (m, 428H, -CH ₂ CH ₂ CH ₂ -derived from polycaprolactone, -CH ₂ CH(COO-)-derived from polyacrylic acid), 2.27 (m, 214H, -CH ₂ C (=O)O-derived from polycaprolactone, _-CH2CH (COO-)-derived from polyacrylic acid) _, 3.51 (s, 456H, _-OCH2CH2O -derived from polyethylene glycol), 3.98 (t, 114H, -C(=O) _OCH2 -derived from polycaprolactone), 12.24. (s, 30H, _-CH2CH (COOH)-derived from polyacrylic acid).

Example 5: Preparation of degradable amphiphilic triblock copolymer micelles

(5.a.) Preparation of degradable amphiphilic triblock copolymer micelles A

Dissolve 10.0 mg of the degradable amphiphilic tri-block copolymer A obtained in Example 1 in 1 mL of acetone, slowly add 4 mL of deionized water (the dropping rate is 0.3 mL/min), stir for 10 hours, and put the solution into In the dialysis bag [molecular weight cut off (MWCO) = 3500], dialyze with deionized water for 48 hours, change the water once every 4 hours, and obtain the degradable amphiphilic triblock copolymer micelles A with a concentration of about 2g/L. aqueous solution.

The degradable amphiphilic triblock copolymer micelle A has a core-shell structure, and its average particle size is 26 nanometers; wherein: the outer shell of the micelle is polyethylene glycol, the inner core is polycaprolactone, and polyacrylic acid is located in the inner core The outer surface. Fig. 6(A) is the radius distribution curve of the degradable amphiphilic triblock copolymer micelle A measured by dynamic light scattering, and its average radius is 13 nm.

(5.b.) Preparation of degradable amphiphilic triblock copolymer micelles B

Dissolve 10.0 mg of the degradable amphiphilic tri-block copolymer B obtained in Example 2 in 0.5 mL of tetrahydrofuran, slowly add 5 mL of deionized water dropwise (the drop rate is 0.3 mL/min), stir for 10 hours, and fill the solution with Put it into a dialysis bag [molecular weight cut-off (MWCO)=3500], dialyze with deionized water for 48 hours, change the water once every 4 hours, and obtain degradable amphiphilic triblock copolymer micelles B with a concentration of about 2g/L of aqueous solution.

The degradable amphiphilic triblock copolymer micelle B has a core-shell structure, and its average particle size is 23.6 nanometers; wherein: the outer shell of the micelle is polyethylene glycol, the inner core is polycaprolactone, and polyacrylic acid is located in the inner core The outer surface. Fig. 6(B) is the radius distribution curve of the degradable amphiphilic triblock copolymer micelle B measured by dynamic light scattering, and its average radius is 11.8 nm.

(5.c.) Preparation of degradable amphiphilic triblock copolymer micelles C

Dissolve 10.0 mg of the degradable amphiphilic tri-block copolymer C obtained in Example 3 in 1 mL of dimethylformamide, slowly drop the dimethyl formamide solution of the above-mentioned amphiphilic tri-block copolymer C into 100 mL In deionized water (a drop rate of 0.3 mL/min), dimethylformamide was removed by ultrafiltration, and after concentration, an aqueous solution of degradable amphiphilic triblock copolymer micelles C with a concentration of about 2 g/L was obtained.

The degradable amphiphilic triblock copolymer micelle C has a core-shell structure, and its average particle size is 30 nanometers; wherein: the outer shell of the micelle is polyethylene glycol, the inner core is polycaprolactone, and polyacrylic acid is located in the inner core The outer surface. Fig. 6(C) is the radius distribution curve of the degradable amphiphilic triblock copolymer micelle C measured by dynamic light scattering, and its average radius is 15 nm.

(5.d.) Preparation of degradable amphiphilic triblock copolymer micelles D

Dissolve 10.0 mg of the degradable amphiphilic tri-block copolymer D obtained in Example 4 in 10 mL of tetrahydrofuran, slowly add 20 mL of deionized water (the drop rate is 0.3 mL/min), stir for 10 hours, and put the solution into [Molecular weight cut-off (MWCO) = 3500] in the dialysis bag, dialyze with deionized water for 48 hours, change the water every 4 hours, and obtain degradable amphiphilic triblock copolymer micelles with a concentration of about 2g/L after concentration D in water.

The degradable amphiphilic triblock copolymer micelle D has a core-shell structure, and its average particle diameter is 184 nanometers; wherein: the outer shell of the micelle is polyethylene glycol, the inner core is polycaprolactone, and polyacrylic acid is located in the inner core The outer surface. Fig. 6(D) is the radius distribution curve of the degradable amphiphilic triblock copolymer micelle D determined by dynamic light scattering, and its average radius value is 92 nm.

The process of carrying out the dynamic light scattering test on the aqueous solution of the above-mentioned degradable amphiphilic tri-block copolymer micelles is as follows: the aqueous solution of the above-mentioned degradable amphiphilic tri-block copolymer micelles is respectively sampled and passed through a filter membrane of 0.45 μm Carry out purification dust removal, utilize laser light scattering instrument (model is ALV/DLS/SLS-5022F, German ALV company produces) at 25 ℃, 90 ° angle measurement degradable amphiphilic triblock copolymer micelle in aqueous solution Hydrodynamic radius.

Embodiment 6: Preparation of anti-tumor nanoparticles

(6.a.) Preparation of anti-tumor nanoparticles A

Dissolve 10.0 mg of the degradable amphiphilic tri-block copolymer A obtained in Example 1 (the number average molecular weight is 10100, wherein COO ^- is 0.025 mmol) and 2.0 mg of doxorubicin are dissolved in 0.1 mL of dimethylformamide and 1 mL of Slowly add 4 mL of deionized water (dropping rate: 0.3 mL/min) into the THF mixed solution, stir for 10 hours, remove THF by rotary evaporation, put the solution into a dialysis bag [molecular weight cut-off (MWCO) = 3500], and use Ionized water dialysis for 48 hours, changing the water once every 4 hours, the intermediate product of the anti-tumor nanoparticles A loaded with doxorubicin at a concentration of about 3g/L was obtained; 4.2 mg of silver nitrate (molecular weight: 170, 0.025 mmol) was suspended in 2 mL of deionized water and stirred for 24 hours at room temperature in the dark, and filtered to remove silver chloride to obtain a hydrated cisplatin solution; Mix the intermediate product of Particle A with cisplatin hydrate solution (Pt/COO ^- is 1), stir at 37°C in the dark for 72 hours, put it into a dialysis bag [molecular weight cut-off (MWCO) = 3500], and dialyze with deionized water For 24 hours, the water was changed every 4 hours to obtain an aqueous solution of anti-tumor nanoparticles A with a concentration of about 2 g/L.

The anti-tumor nanoparticle A has a core-shell structure, and its average particle size is 30 nanometers; wherein: the outer shell of the anti-tumor nanoparticle A is polyethylene glycol; the inner core is polycaprolactone, and the polycaprolactone is loaded with A Mycin; polyacrylic acid is located on the outer surface of the inner core and supports cisplatin through the coordination complexation between carboxylate and platinum in cisplatin. Fig. 8(A) is the radius distribution curve of the anti-tumor nanoparticle A measured by dynamic light scattering, and its average radius value is 15 nanometers. The mass of doxorubicin loaded by anti-tumor nanoparticles A accounts for 6.3% of the mass of anti-tumor nanoparticles A; the platinum (Pt) of cisplatin loaded by anti-tumor nanoparticles A is relatively The molar ratio (Pt/COO ^- ) of the carboxylate group in the substance A was 0.2.

(6.b.) Preparation of anti-tumor nanoparticles B

10.0 mg of the degradable amphiphilic tri-block copolymer B obtained in Example 2 (the number average molecular weight is 5500, wherein COO ^- is 0.045 mmol) and 1.0 mg of doxorubicin are dissolved in 0.1 mL of dimethylformamide and 1 mL of Slowly add 10 mL of deionized water (dropping rate: 0.3 mL/min) into the THF mixed solution, stir for 10 hours, remove dimethylformamide, THF and unloaded doxorubicin by ultrafiltration, and concentrate to obtain the concentration About 3g/L of the intermediate product of anti-tumor nanoparticles B loaded with doxorubicin; the intermediate product of anti-tumor nanoparticles B loaded with doxorubicin was mixed with 6.8mg cisplatin (0.023mmol) (Pt/COO ^- is 0.5), and after 72 hours of stirring in the dark at 25° C., the unloaded cisplatin was removed by ultrafiltration to obtain an aqueous solution of anti-tumor nanoparticles B with a concentration of about 2 g/L.

The anti-tumor nanoparticle B has a core-shell structure, and its average particle diameter is 25 nanometers; wherein: the outer shell of the anti-tumor nanoparticle B is polyethylene glycol; Mycin; polyacrylic acid is located on the outer surface of the inner core and supports cisplatin through the coordination complexation between carboxylate and platinum in cisplatin. Fig. 8(B) is the radius distribution curve of anti-tumor nanoparticles B measured by dynamic light scattering, and its average radius value is 12.5 nm. The mass of doxorubicin loaded on anti-tumor nanoparticles B accounts for 1% of the mass of anti-tumor nanoparticles A; the platinum (Pt) of cisplatin loaded on anti-tumor nanoparticles B is relatively The molar ratio (Pt/COO ^- ) of the carboxylate group in the substance B was 0.1.

(6.c.) Preparation of anti-tumor nanoparticles C

10.0 mg of degradable amphiphilic tri-block copolymer C (the number average molecular weight is 9300, wherein COO ^- is 0.0086 mmol) and 5.0 mg of doxorubicin are dissolved in 1 mL of dimethylformamide, slowly Add 15 mL of deionized water dropwise (the dropping rate is 0.3 mL/min), stir for 10 hours, put the above solution into a dialysis bag [molecular weight cut-off (MWCO) = 3500], dialyze with deionized water for 48 hours, change every 4 hours Water once, and concentrated to obtain the intermediate product of anti-tumor nanoparticles C loaded with doxorubicin at a concentration of about 3g/L; 5.2mg cisplatin (0.017mmol) and 2.9mg silver nitrate (0.017mmol) were suspended in 2mL to remove Stir in deionized water for 24 hours at room temperature and avoid light, filter to remove silver chloride, and obtain cisplatin hydrate solution; mix the intermediate product of anti-tumor nanoparticles C loaded with doxorubicin and cisplatin hydrate solution (Pt/ ^COO- For 2), stir in the dark at 50°C for 96 hours, put it into a dialysis bag [molecular weight cut-off (MWCO) = 3500], dialyze with deionized water for 24 hours, change the water every 4 hours, and obtain a concentration of about 2g/ Aqueous solution of antitumor nanoparticles C in L.

The anti-tumor nanoparticle C has a core-shell structure, and its average particle diameter is 54 nanometers; wherein: the outer shell of the anti-tumor nanoparticle C is polyethylene glycol; Mycin; polyacrylic acid is located on the outer surface of the inner core and supports cisplatin through the coordination complexation between carboxylate and platinum in cisplatin. FIG. 8(C) is the radius distribution curve of the anti-tumor nanoparticle C measured by dynamic light scattering, and its average radius value is 27 nanometers. The mass of doxorubicin loaded on anti-tumor nanoparticles C accounts for 10% of the mass of anti-tumor nanoparticles C; the platinum of cisplatin (Pt) loaded on anti-tumor nanoparticles C is relatively The carboxylate molar ratio (Pt/COO ⁻ ) value of the substance C was 0.9.

(6.d.) Preparation of anti-tumor nanoparticles D

10.0 mg of the degradable amphiphilic tri-block copolymer D (the number average molecular weight is 22800, wherein COO ^- is 0.013 mmol) and 10.0 mg of doxorubicin are dissolved in 1 mL of dimethylformamide, slowly Add 20 mL of deionized water dropwise (the dropping rate is 0.3 mL/min), stir for 10 hours, put the above solution into a dialysis bag [molecular weight cut-off (MWCO) = 3500], dialyze with deionized water for 48 hours, change every 4 hours Water once, after concentrating, obtain the intermediate product of the anti-tumor nanoparticle D loaded with doxorubicin at a concentration of about 3g/L; the intermediate product of the antitumor nanoparticle D loaded with doxorubicin and 0.4mg cisplatin (0.0013 mmol) mixed (Pt/COO ^- is 0.1), stirred at 20°C in the dark for 48 hours, then put into a dialysis bag [molecular weight cut-off (MWCO) = 3500], dialyzed with deionized water for 24 hours, changing the water every 4 hours Once, an aqueous solution of anti-tumor nanoparticles D with a concentration of about 2 g/L was obtained.

The anti-tumor nanoparticle D has a core-shell structure, and its average particle diameter is 176 nanometers; wherein: the outer shell of the anti-tumor nanoparticle D is polyethylene glycol; Mycin; polyacrylic acid is located on the outer surface of the inner core and supports cisplatin through the coordination complexation between carboxylate and platinum in cisplatin. Fig. 8(D) is the radius distribution curve of anti-tumor nanoparticles D measured by dynamic light scattering, and the average radius value is 88 nm. The mass of doxorubicin loaded on anti-tumor nanoparticles D accounts for 50% of the mass of anti-tumor nanoparticles D; the platinum (Pt) of cisplatin loaded on anti-tumor nanoparticles D is relatively The molar ratio (Pt/COO ^- ) of the carboxylate group of the substance D was 0.05.

The process of carrying out the dynamic light scattering test on the aqueous solution of the above-mentioned anti-tumor nanoparticles is as follows: the aqueous solution of the above-mentioned anti-tumor particles is respectively sampled and purified and dedusted through a filter membrane of 0.45 μm, and the laser light scattering instrument (model is ALV/DLS/SLS -5022F, produced by ALV Company in Germany) at 25°C and at an angle of 90° to measure the hydrodynamic radius of anti-tumor nanoparticles in aqueous solution.

Example 7: Release of antitumor drugs doxorubicin and cisplatin from antitumor nanoparticles A

At 37°C, use dialysis method [dialysis membrane (MWCO) = 3500] to evaluate the solution in 10mM PBS (pH = 7.4) containing 160mM NaCl (simulating the acidity, alkalinity and salt content of human body fluids in mammals) , the release behavior of anti-tumor drugs doxorubicin and cisplatin from anti-tumor nanoparticles A described in Example (6.a). The external fluid of the dialysis bag was sampled at specific time intervals, and the concentration of doxorubicin and cisplatin were measured by ultraviolet spectrophotometer.

Fig. 9(A) is the release curve of doxorubicin, and the release rate of doxorubicin reaches over 80% within the detection time of 80 hours. Fig. 9(B) is the release curve of cisplatin, and the release rate of cisplatin reaches over 65% within 250 hours of testing. The results show that most of the antitumor drugs can be released in the buffer solution simulating the in vivo environment and the sustained release of the drugs is realized.

Example 8: Biodegradability of degradable amphiphilic triblock copolymer micelles A

Put 60 μL of the acetone solution (mass concentration of 0.005 g/L) of the fluorescent probe molecule pyrene into a glass bottle, and add the degradable amphiphilic triblock copolymerization solution described in Example (5.a) after the acetone evaporates. The aqueous solution of object micelle A 3mL, sonicate 0.5 hour and stand still for 24 hours. Put the above solution in a water bath at 37°C, add lipase Lipase PS to make the enzyme concentration in the solution 0.01g/L, measure the fluorescence intensity of this aqueous solution at specific time intervals (excitation wavelength is 335nm, detection wavelength is 350-500nm) .

Figure 10 is the degradation experiment result of the degradable amphiphilic triblock copolymer micelle A: under the action of lipase Lipase PS, the polycaprolactone segment of A in the degradable amphiphilic triblock copolymer micelle Degradation due to the hydrolytic cleavage of the ester bond results in the rupture of the core of the degradable amphiphilic triblock copolymer micelle A, and the fluorescent probe pyrene wrapped in the core of the degradable amphiphilic triblock copolymer micelle A Released into the water, the fluorescence is quenched by small molecules such as oxygen in the water and the fluorescence detection intensity is reduced. As shown in FIG. 10 , the fluorescence intensity of the fluorescent probe pyrene decreased from 142 at 0 minutes to 112 at 80 minutes.

Example 9: Cytotoxicity of degradable amphiphilic triblock copolymer micelles A and antitumor nanoparticles A

Toxicity of degradable amphiphilic triblock copolymer micelles A and antitumor nanoparticles A on human bladder cancer cells was evaluated by MTT assay. Human bladder cancer cells were cultured in 50 μL RPMI1640 medium containing 10% fetal bovine serum in a 96-well plate (3000 cells per well), and degradable amphiphilic triblock copolymer micelles A or anti- Tumor nanoparticles A made the concentration of each 150mg/L, after the cells were incubated at 37°C in a humid environment with a volume concentration of 5% carbon dioxide for 48 hours or 72 hours, MTT solution was added, and after 4 hours of incubation, it was replaced by dimethyl The sulfoxide solution was used to determine the cell viability (%) by measuring the absorbance at 570 nm with a microplate reader (model MULTISCAN MK-III, produced by Thermoelectric Corporation of America) and the results are summarized in Table 1.

Table 1

Within the time involved in Example 9, the degradable amphiphilic triblock copolymer micelle A has no inhibitory performance on the growth of human bladder cancer cells, indicating that the degradable amphiphilic triblock copolymer micelle A has no inhibitory effect on human bladder cancer. The cells are avirulent or substantially avirulent. After loading anti-tumor drugs, anti-tumor nanoparticles A showed high inhibitory effect on human bladder cancer cells.

Industrial applicability:

The anti-tumor nanoparticles of the present invention can be loaded with one or more anti-tumor drugs through hydrophobic interaction and coordination complexation, have biodegradability, and can be used in chemotherapy of human tumors.

Claims (9)

1. A degradable amphiphilic triblock copolymer micelle, which is a degradable amphiphilic triblock copolymer composed of polyethylene glycol segment, polycaprolactone segment and polyacrylic acid segment Obtained after self-assembly in an aqueous solution, it is characterized in that: the degradable amphiphilic triblock copolymer micelle has a core-shell structure, and its average particle diameter is 20-200 nanometers; wherein: the shell of the micelle is polyethylene Diol, the core is polycaprolactone, and polyacrylic acid is located on the outer surface of the core; The degradable amphiphilic tri-block copolymer has the following structural formula (A):

In the above formula, R ₁ is derived from the terminal structure of the polyethylene glycol segment, R ₁ is a hydrogen atom, or one of straight-chain or branched C _1-12 alkyl groups; L is a linking group, and the linking The base is derived from a residue after reaction of one of the halogenated carboxylic acid, the halogenated acid chloride or the halogenated acid bromide with the polyethylene glycol-block-polycaprolactone diblock copolymer; R ₂ is halogen, and the Halogen is obtained by reacting one of halogenated carboxylic acid, halogenated acid chloride or halogenated acid bromide with polyethylene glycol-block-polycaprolactone diblock copolymer; m is an integer of 45-120, n is an integer of 5-60, y is an integer of 10-100; x is a hydrolysis rate, and the hydrolysis rate is 30%-100%. 2. degradable amphiphilic triblock copolymer micelle according to claim 1 is characterized in that: described C _1-12 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, decyl or undecyl; The residue is -CO-C(CH ₃ ) ₂ -, -CO-CH(CH ₃ )-, -CO-C ₆ H ₄ -CH ₂ - or -CO-CH(CN)-; The halogen is Cl, Br or I. 3. a preparation method of the degradable amphiphilic triblock copolymer micelle described in claim 1 or 2, is characterized in that: the degradable amphiphilic triblock copolymer is dissolved in an organic solvent to obtain A solution containing a degradable amphiphilic triblock copolymer, drop a solution containing a degradable amphiphilic triblock copolymer into water, or drop water into a solution containing a degradable amphiphilic triblock copolymer , wherein the volume ratio of water to organic solvent is greater than 1; then the organic solvent is removed by ultrafiltration or dialysis to obtain degradable amphiphilic triblock copolymer micelles. 4. The preparation method according to claim 3, characterized in that: the organic solvent is at least one selected from the group consisting of tetrahydrofuran, acetone, and dimethylformamide. 5. The preparation method according to claim 3, characterized in that: the degradable amphiphilic tri-block copolymer is prepared by the following method: (1) Mixing polyethylene glycol with R ₁ at one end and hydroxyl at the other end and caprolactone monomer at a molar ratio of 1:5-200, and then dissolving the mixture as a reactant in dry toluene, Add catalyst amount of 1,5,7-triazabicyclo-[4.4.0]dec-5-ene, react at room temperature; or The mixture is used as a reactant, and the catalyst stannous octoate is added to the reactant, wherein the amount of stannous octoate added is that the molar ratio of polyethylene glycol to stannous octoate is 10-30; at a reaction temperature of 100-150°C reaction; The product obtained by the reaction is precipitated in a precipitant and dried to obtain a polyethylene glycol-block-polycaprolactone diblock copolymer; (2) Mix the polyethylene glycol-block-polycaprolactone diblock copolymer obtained in step (1) with the halogenated compound in a molar ratio of 1:1-10, and then dissolve the mixture as a reactant in a dry In dichloromethane, dry triethylamine is added as a catalyst, wherein the molar ratio of triethylamine to halide is 1, react at room temperature, filter to remove the salt generated by the reaction, precipitate in a precipitant and dry to obtain poly Ethylene glycol-block-polycaprolactone diblock copolymer initiator; The halogenated compound is selected from α-halogenated isobutyric acid, α-halogenated isobutyryl chloride, α-halogenated isobutyryl bromide, α-halogenated isopropionic acid, α-halogenated isopropionyl chloride, α- Halogenated isopropionyl bromide, Halogenated methylbenzoic acid, Halogenated methylbenzoyl chloride, Halogenated methylbenzoyl bromide, α-halogenated cyanoacetic acid, α-halogenated cyanoacetyl chloride, α-halogenated cyanoacetyl bromide one of (3) the polyethylene glycol-block-polycaprolactone diblock copolymer initiator that step (2) obtains is mixed with the tert-butyl ester monomer in the ratio of 1: 10-300 with molar ratio, then The mixture is dissolved in dry anisole or acetone as a reactant, ketone bromide and pentamethyldiethylenetriamine are added, and the polyethylene glycol-block-polycaprolactone diblock copolymer initiates Reagent: cuprous bromide: the molar ratio of pentamethyldiethylenetriamine is 2:1:1, at any constant temperature between room temperature and 120°C, and react under anaerobic conditions; the reaction obtained The product is precipitated in a precipitant and dried to obtain a polyethylene glycol-block-polycaprolactone-block-polyacrylate tert-butyl triblock copolymer after the column of overbasic alumina; (4) the polyethylene glycol-block-polycaprolactone-block-polyacrylate tert-butyl ester triblock copolymer obtained in step (3) is dissolved in methylene chloride, and trifluoroacetic acid is added as a catalyst, Wherein the molar ratio of trifluoroacetic acid and the tert-butyl group of the poly(tert-butyl acrylate) in polyethylene glycol-block-polycaprolactone-block-polyacrylate tert-butyl acrylate triblock copolymer is 3-10, React at room temperature, precipitate the product obtained after the reaction in a precipitant and dry to obtain the degradable amphiphilic triblock copolymer polyethylene glycol-block-polycaprolactone-block of formula (A) in claim 1 segment - polyacrylic acid; The precipitating agent is selected from ether, n-hexane and petroleum ether. 6. the purposes of a kind of degradable amphiphilic triblock copolymer micelle described in claim 1 or 2 is characterized in that: described degradable amphiphilic triblock copolymer micelle uses as drug carrier . 7. purposes according to claim 6, it is characterized in that: described as drug carrier is by the hydrophobic effect load of the hydrophobic biodegradable polycaprolactone segment of degradable amphiphilic triblock copolymer Hydrophobic anti-tumor drugs, and then through the coordination and complexation of the carboxylate group of the polyacrylic acid segment and the platinum in the cis-platinum (II) anti-tumor drugs to load the cis-platinum (II) anti-tumor drugs to form anti-tumor nanoparticles Particles; the anti-tumor nanoparticles have a core-shell structure, and their average particle diameter is 20-200 nanometers; wherein: the outer shell of the anti-tumor nanoparticles is polyethylene glycol; the inner core is a polymer loaded with hydrophobic anti-tumor drugs through hydrophobic interaction Caprolactone; polyacrylic acid loaded with cis-platinum (II) antitumor drugs through coordination and complexation is located on the outer surface of the inner core. 8. The use according to claim 7, characterized in that: the mass of the hydrophobic anti-tumor drug loaded on the anti-tumor nanoparticles accounts for 1%-50% of the mass of the anti-tumor nanoparticles; The molar ratio of the platinum of the cis-platinum (II) class antitumor drug loaded on the nanoparticles relative to the carboxylate radical of the degradable amphiphilic triblock copolymer is 0.05-1. 9. purposes according to claim 7, it is characterized in that: the preparation process of the hydrophobic effect of described polycaprolactone chain segment loading hydrophobic antitumor drug is the degradable amphiphilic tri-block copolymer and Hydrophobic antitumor drugs are dissolved in organic solvents at the same time to obtain a mixed solution containing degradable amphiphilic triblock copolymers and hydrophobic antitumor drugs. A mixed solution of tumor drugs is dropped into water, or water is dropped into a mixed solution containing degradable amphiphilic tri-block copolymers and hydrophobic anti-tumor drugs, wherein the volume ratio of water to organic solvent is greater than 1; Filtration or dialysis to remove organic solvents and unloaded hydrophobic antitumor drugs to obtain intermediates of antitumor nanoparticles loaded with hydrophobic antitumor drugs; wherein the organic solvent is selected from tetrahydrofuran, acetone, dimethylformamide at least one of the group consisting of; The preparation process of loading cis-platinum (II) anti-tumor drugs through the coordination and complexation of the carboxylate of the polyacrylic acid segment and the platinum in the cis-platinum (II) anti-tumor drugs is that the above-mentioned loaded Intermediate products of anti-tumor nanoparticles with hydrophobic anti-tumor drugs and cis-platinum (II) anti-tumor drugs are placed in water as reactants, wherein: platinum in cis-platinum (II) anti-tumor drugs is compared to polyacrylic acid The feeding molar ratio of carboxylate groups in the chain segment is 0.1-2; react at any constant temperature between 20-50°C for 24-96 hours, and then remove unsupported cis-platinum (II) by ultrafiltration or dialysis ) class of antitumor drugs to obtain antitumor nanoparticles.

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